PROJECT SUMMARY: Protein ubiquitination functions in many biological processes. Histone ubiquitination, an epigenetic mark, governs DNA-related processes though chromatin structure and transactions. Misregulation in ubiquitination and epigenetic mechanisms are linked to numerous inherited and acquired diseases including diabetes, cancers and neurological disorders. Histone H2B monoubiquitination (H2Bub1) regulates gene transcription from yeast to humans. This proposal focuses on the evolutionarily conserved budding yeast H2B ubiquitin-conjugating complex (HUC) comprised of Rad6 (E2 conjugase), a homodimer of Bre1 (E3 ligase) and Lge1 (an accessory protein). Assembly, recruitment, and regulation of HUC complex are not fully understood. Using extensive functional, biochemical and structural studies including a preliminary crystal structure for Rad6-Bre1 sub-complex, we have identified multiple subunit interfaces that are distinct to the HUC complex compared to other ubiquitin-conjugating complexes including a novel interface between a non-catalytic region of Rad6 and a non-RING domain region of Bre1, an asymmetric conformational state of the Bre1 dimer, and Lge1 binding near the Bre1 RING domain. We will use X-ray crystallography to visualize these distinctive subunit contacts in Aim 1. Lge1, an essential but uncharacterized accessory protein, binds chromatin factors and Bre1 and is expendable for Rad6-Bre1's K63-linked polyubiquitination activity, prompting the intriguing hypotheses that Lge1 recruits the HUC complex to relevant sites on chromatin and restricts its activity to monoubiquitination, which will also be tested in Aim 1 using genomics to determine the genome-wide occupancy of the HUC complex and a biochemistry-proteomics combination to measure mono versus poly ubiquitination. Aim 2 builds on our confirmation of in vivo phosphorylation of serine-120 near Rad6's catalytic cleft. Our preliminary structure for Rad6 with the phosphomimetic aspartate substitution suggests that S120 phosphorylation is a molecular switch that `closes' down the active site to inhibit monoubiquitination but allow polyubiquitination. This will be tested by determining the structure of S120-phosphorylated Rad6. Our preliminary data also involves the discovery that the cell wall integrity pathway kinase Mpk1 phosphorylates Rad6. This motivates the model that this modification activates Rad6 to polyubiquitinate substrates during transcription initiation and in upstream signaling events, and that subsequent dephosphorylation makes Rad6 a H2B monoubiquitinase during transcription elongation promoting gene expression. This innovative regulatory switch theory will be tested in Aim 2 using a multipronged approach to determine genome-wide occupancies, transciptomes and ubiquitomes regulated by Rad6 and Rad6S120phos. In summary, our multidisciplinary studies will advance the ubiquitination and epigenetics fields by addressing fundamentally important functional and mechanistic questions regarding Rad6 and the HUC complex. Given the evolutionary conservation, our studies are applicable to the epigenetic and ubiquitination mechanisms in metazoans including humans.